1994 Volume 116 Issue 5 Pages 1139-1145
Twenty-two mutants between βGlu-161 and βLys-201 of Escherichia coli H+-ATPase β subunit could grow by oxidative phosphorylation, but 11 other such mutants, βGlu-181→Gln, Asp, Asn, Thr, Ser, Ala, or Lys and βArg-182→Lys, Ala, Glu, or Gln, could not. The βAsp-181, β3Lys-182, and other defective mutants had 1.4, 1, and <0.1%, respectively, of the wild-type membrane ATPase activity. Partially purified F1-ATPases from all mutants at positions 181 and 182, except for the, βAsp-181 and βLys-182 mutants, showed very low unisite catalysis. Purified F1-ATPases of the β3Gln-181 and βAla-181 mutants showed no multisite (or steady state) catalysis and slow unisite catalysis (≤1% of that of the wild type): their defects could be attributed to decreased catalytic rates (low k+2 and k-2). Changes of the k+2 and k-2 values in the, βAsp-181 enzyme, which showed detectable multi- and unisite catalysis, were less marked (27 and 21%, respectively, of wild-type rates). The β3Gln-182 enzyme showed defective catalysis (≤0.1% of the multi- and _??_1% of the unisite catalyses of the wild type), whereas the βLys-182 enzyme showed 1 and 85% of the wild-type multisite and unisite catalytic rates, respectively. βLys-182 had wild-type values of k+2 and k-2, but βGln-182 had k+2 about 10-fold lower than that of wild type. The position 181 and 182 mutant enzymes had significantly increased Kd (k-1, / k+1) values, reflecting decreased substrate binding. These results suggest that βGlu-181 and βArg-182 are essential for substrate binding, although mutations with conservative substitutions at these positions do not have drastic effects. This study also indicates the importance of the conserved Gly-Glu-Arg (GER) sequence (β180-β182).
